Interpretive Summary: Aflatoxins, the toxic and carcinogenic compounds found in some foods and feeds that are contaminated by the mold Aspergillus flavus, are first made from a precursor molecule. The precursor molecule is formed by a specialized enzyme that takes small organic acids and joins them together over and over again until the pocket of the enzyme is filled. It then “spits out” the finished aflatoxin precursor. One of the organic acids that is required for aflatoxin production is a six carbon fatty acid. The other is a two carbon fatty acid. The specialized enzyme that does this is called a polyketide synthase. It has many regions that carry out the individual steps of the process. Critical to the process is a region near the beginning of the protein called the starter unit domain. In the polyketide synthase that makes aflatoxin, the starter unit accepts the six carbon fatty acid, but in most others, the starter unit is a one or two carbon organic acid. This paper demonstrated conclusively that all functional polyketide synthases have a starter unit and the nature of the critical amino acids in the domain dictate how big an acid the domain can accept. This study better defines just what happens at the beginning of aflatoxin biosynthesis and could be useful for developing compounds to inhibit aflatoxin production without causing inhibition of plant growth or without being toxic to humans and animals.

Technical Abstract:
Polyketide natural products are found in bacteria, fungi, and plants. They contribute disproportionately to the arsenal of clinically used agents, stimulating investigation of their biosynthetic pathways for purposes of improved production and metabolic engineering. Polyketides isolated from fungi are typically derived from “iterative” Type I (multidomainal) polyketide synthases (PKSs), where individual catalytic domains are fused into single large proteins, and reused a fixed number of times in the “programmed” synthesis of a given product. The best known examples of iterative Type I enzymes are the yeast and animal fatty acid synthases (FASs). More than 50 distinct chemical steps take place without release of an intermediate, and most domains are used at least seven times in the synthesis of a single palmitoyl chain (C16). FASs are highly evolved to carry out each elongating reaction one time for every two-carbon homologation to accomplish synthesis of the fully reduced fatty acid. Iterative Type I PKSs utilize many of the same catalytic domains, but the “programming” of these enzymes to select starter units, initiate synthesis, control chain length, oxidation state, and cyclization chemistry are central unanswered questions in natural products chemistry. The first of these processes is addressed in this paper.
Multistep animal fatty acid biosynthesis is initiated by acetyl-CoA, which is brought onto the enzyme by a bifunctional acyl transferase, malonyl-CoA:acyl carrier protein (ACP) transacylase (MAT), which primarily shuttles units of malonyl-CoA for each successive two-carbon omologation leading to palmitate. Alternatively, in fungi an A6B6 heterododecameric FAS 21 complex harbors a specific acetyl transacylase for starter unit introduction. In classical precursor incorporation experiments with fatty acids and fungal natural products, it was commonly observed that an acetate starter unit would bear a higher specific incorporation of radiolabel from [14C]-acetate than the rest of the labeled sites in the molecule, in keeping with the intermediary conversion of acetyl-CoA to malonyl-CoA for each chain extension [6]. Conversely, if the complementary incorporation of [14C]-malonate were examined, the starter 3
carbons would often be distinguishably more weakly labeled than the remaining portions of the polyketide metabolite. This general pattern came to be known as the “starter unit effect.” In this paper we demonstrate for the broad class of “nonreducing” iterative fungal PKSs that a specialized domain has evolved to load starter units to initiate polyketide synthesis, while the MAT domains present in these systems are restricted largely to malonyl transfer.
The UMA algorithm combines primary sequence similarity, predicted secondary structure, and local hydrophobicity among members of a family of related proteins to identify probable linker regions between functional domains. Application of this algorithm to PksA (GenBank accession no. AY371490), the iterative Type I PKS central to the biosynthesis of the mycotoxin aflatoxin, unveiled two previously unrecognized, but clearly resolved domains. (Figure 1A) A central domain (~350 amino acids) was suggested to be involved in facilitating correct cyclization chemistry of a hypothetical poly B-keto intermediate. We recently established that the large N-terminal domain is a starter unit:ACP transacylase (SAT) that selectively introduced hexanoyl starter units onto the PksA ACP to prime norsolorinic acid (1) biosynthesis[9]. Search of genome databases revealed that SAT domains were widespread among known and 18
apparent nonreducing fungal PKSs. PksA is unusual among fungal PKSs in that the rare hexanoyl starter unit is supplied by a dedicated yeast-like pair of FAS subunits, HexA and HexB. The three wild-type enzymes have been co-purified from Aspergillus parasiticus and are thought to form anA2B2Y2 dimeric heterotrimer distinct from the canonical yeast/fungal A6B6 FAS organization] . The vast majority of fungal polyketides, however, are synthesized from acetyl-CoA and malonyl-CoA. These enzymes require no specialized apparatus to prepare a starter unit when these rudimentary building blocks are already available in the cell. In this paper, we compare four related nonreducing iterative PKSs (none having an associated FAS pair) and demonstrate that the SAT domain in each selectively transfers acetyl-CoA in preference to higher CoA homologs or malonyl-CoA.